Apoptosis is initiated via assembly of multimeric caspase-activating signaling complexes. One such important complex is the PIDDosome for caspase-2 activation, which contains the p53-inducible protein PIDD, RAIDD and caspase-2. Caspase-2 is an initiator caspase and the most evolutionarily conserved caspase. In response to DNA damage and other cellular stress, caspase-2 acts upstream of the mitochondria to mediate cell death by inducing Bid cleavage, Bax translocation and cytochrome c release. Like other caspase activating complexes and many kinase activating complexes, assembly of the PIDDosome is dependent on proteins of the death domain (DD) superfamily. The DD superfamily comprises four subfamilies, the DD subfamily, the caspase recruitment domain (CARD) subfamily, the death effector domain (DED) subfamily and the pyrin domain (PYD) subfamily and shares the similar six helical bundle fold. Full length PIDD contains 910 residues with seven leucine rich repeats (LRRs), two ZU-5 domains and a C-terminal DD. RAIDD contains an N-terminal CARD and a C-terminal DD. Caspase-2 has a CARD prodomain at its N-terminal end and a caspase domain at its C-terminal end. The PIDDosome is assembled via a DD: DD interaction between RAIDD and PIDD and a CARD: CARD interaction between RAIDD and caspase-2. PIDD DD is not only essential for the activation of caspase-2 it can also interact with the DD of RIP1, a kinase implicated in the activation of NF-B. PIDD acts as a molecular switch, controlling the balance between life and death. No structural information is available on the PIDDosome. In this application, we propose a series of structural and biological studies to elucidate the molecular basis of PIDDosome formation. The studies will not only contribute to our understanding of PIDDosome formation but also DD superfamily interactions in other assemblies in general. PUBLIC HEALTH RELEVANCE: Apoptosis plays an important role in human physiology. Deregulation of apoptosis is related to many human diseases. The current application seeks to address a critical question in apoptosis regulation and has a strong implication in understanding and treatment of apoptosis-related human diseases.